Molecular imaging has been actively conducted which performs imaging for visualization of cells, particularly, living cells, or imaging for visualization targeting molecules in living bodies to reveal molecular kinetics, intermolecular interaction, and molecular positional information, leading to the elucidation of the mechanisms of life science or drug discovery screening. Particularly, researches have also been actively made to detect cancer cells or cancer sites by visualizing abnormal cells, for example, cancer cells.
The group of the present inventors have proposed a method using green fluorescence-emitting 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) in which a N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino group is bound as a fluorophore to the 2-position of D-deoxyglucose, as a method that can be used in research on the dynamic uptake process of D-glucose by live cells, and have demonstrated the usefulness of the method using various mammalian cells (Non Patent Literature 1).
This method utilizes 2-NBDG's property of being selectively taken into live cells and can quantitatively determine dynamic activity as to the cellular uptake of D-glucose by monitoring change in fluorescence intensity caused by the uptake. Therefore, this method is highly regarded by researchers around the world as a breakthrough for studying how organisms take D-glucose into cells and utilize the D-glucose, and is now accepted as a standard protocol indispensable in this research field (Non Patent Literature 2).
In the history of the previous development of fluorescent D-glucose derivatives, 2-NBDG and 6-NBDG which are D-glucose derivatives bound to a green fluorescent group NBD are the only substances that have been internationally accepted as being transported into cells via a glucose transporter GLUT and subjected to various replication studies. Furthermore, 2-NBDG is the only molecule that has been found to be taken into cells and then phosphorylated like FDG that has been used in the PET examination of cancers. Therefore, since long ago, it has been suggested that 2-NBDG can be utilized not only for the purposes of basic science fields but also for application to tumor cell imaging in clinical medicine (Non Patent Literature 3, Patent Literature 1, etc.). Also, many attempts have been made to apply 2-NBDG to the diagnostic imaging of cancers (Non Patent Literatures 4 and 5).
As another compound than 2-NBDG expected to pass through GLUT, the inventors have developed 2-DBDG (2-[N-7-(N′,N′-dimethylaminosulfonyl)benz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose) which is a molecule in which DBD that is an analog of NBD is bound to D-glucose, and have reported the properties of this compound (Patent Literature 2). Even though 2-DBDG has a fluorescence wavelength slightly shifted to the longer wavelength side as compared with 2-NBDG, it largely overlaps with 2-NBDG in terms of spectra. Thus, when 2-DBDG is used with 2-NBDG at the same time, a problem arises for discriminating them from each other.
In fact, D-glucose derivatives bound to various other fluorescent molecules than NBD have also been proposed so far. Particularly, there have been developed a molecule bound to a more highly tissue-permeable probe having the maximum fluorescence in the near-infrared region, a molecule suitable for excitation using a two-photon microscope, a molecule that emits stronger fluorescence than 2-NBDG, and the like. However, because these D-glucose derivative molecules have a much larger molecular size than 2-NBDG, mechanisms underlying their uptake into cells are presumed to be cellular uptake via pathways other than GLUT, for example, uptake by phagocytosis or by internalization in a protein-bound state (Non Patent Literature 6).
In addition to the molecules having a green, red, or near-infrared fluorescence spectrum, fluorescent glucose derivatives have also been reported which have a D-glucose bound to a coumarin derivative molecule having a blue fluorescence spectrum (Esculin, Fraxin, and compounds described in WO2012/70024 (Patent Literature 3)). However, these derivatives are not taken into cells via GLUT. There has been no report on a molecule that is a sugar derivative having, in its molecule, a blue fluorescent molecular group and passes through GLUT.
As for substances in which a quinoline derivative molecule is bound to glucose, G. Wagner et al. (Non Patent Literature 7) have reported the following substances:
and Roman Kimmel et al. (Non Patent Literature 8) have reported the following substance:
However, all of these substances have a structure where a quinoline derivative is bound to the 1-position of glucose either directly or via an oxygen atom. It has not been reported that any of them are taken into cells via GLUT.
Meanwhile, the present inventors have proposed compounds in which 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue) or 3-carboxymethyl-6,8-difluoro-7-hydroxy-4-methylcoumarin (Marina Blue) is bound to glucose via an amide bond, and have filed a patent application of PCT/JP2013/076629.    Patent Literature 1: U.S. Pat. No. 6,989,140 specification    Patent Literature 2: WO2010/16587    Patent Literature 3: WO2012/70024    Patent Literature 4: WO2012/133688    Non Patent Literature 1: Yamada K. et al., J. Biol. Chem. 275: 22278-22283, 2000    Non Patent Literature 2: Yamada K. et al., Nat. Protoc. 2: 753-762, 2007    Non Patent Literature 3: O'Neil et al., Mol. Imaging Biol. 7: 388-392    Non Patent Literature 4: Sheth et al., J. Biomed. Opt. 14: 064014-1-8, 2009    Non Patent Literature 5: Nitin et al., Int. J. Cancer 2009    Non Patent Literature 6: Cheng Z. et al., Bioconjugate Chem. 17: 662-669, 2006    Non Patent Literature 7: G. Wagner, et al., Archiv der Pharmazie, 298 (8), 481-491 (1965)    Non Patent Literature 8: Roman Kimmel, et al., Carbohydr. Res., 345, 768-779 (2010)